Abstract

Inhibition of acetyl-CoA carboxylases (ACCs), a crucial enzyme for fatty acid metabolism, has been shown to promote fatty acid oxidation and reduce body fat in animal models. Therefore, ACCs are attractive targets for structure-based inhibitor design, particularly the carboxyltransferase (CT) domain, which is the primary site for inhibitor interaction. We have cloned, expressed, and purified the CT domain of human ACC2 using baculovirus-mediated insect cell expression system. However, attempts to crystallize the human ACC2 CT domain have not been successful in our hands. Hence, we have been using the available crystal structure of yeast CT domain to design human ACC inhibitors. Unfortunately, as the selectivity of the lead series has increased against the full-length human enzyme, the potency against the yeast enzyme has decreased significantly. This loss of potency against the yeast enzyme correlated with a complete lack of binding of the human-specific compounds to crystals of the yeast CT domain. Here, we address this problem by converting nine key active site residues of the yeast CT domain to the corresponding human residues. The resulting humanized yeast ACC-CT (yCT-H9) protein exhibits biochemical and biophysical properties closer to the human CT domain and binding to human specific compounds. We report high resolution crystal structures of yCT-H9 complexed with inhibitors that show a preference for the human CT domain. These structures offer insights that explain the species selectivity of ACC inhibitors and may guide future drug design programs.

Comparison of yeast (yCT), human (hCT), and humanized (yCT-H9) CT domain active site residues.A, sequence alignment of human ACC2 and yeast ACC-CT domain residues was performed using ClustalW (). Residues in the boxed area are those within 6 Å of the inhibitor (Compound 1) (). The yellow-highlighted residues are the ones that differ between human and yeast at the ligand binding interface. B, shown is molecular surface of the binding site for compound 1 in the yeast CT domain. The yeast active site residues that were mutated to mimic the human ACC-CT domain (nine point mutants as reported in “Experimental Procedures”) are colored in magenta. The structure of Compound 1 (PDB code 1W2X) is depicted as green sticks. C, the active site region of yCT structure (green ribbon) was superimposed on the corresponding structures of hCT (yellow ribbon) and yCT-H9 (cyan ribbon). The binding mode of Compound 1 to yCT (green stick), yCT-H9 (cyan stick), and hCT (yellow stick) was superimposed to show the relative positions.

The inhibition of the CT domain variants.A, shown is a plot of the fluorescence anisotropy of compound 1 as a function of protein concentration. B, the fluorescence anisotropy of compound 1 as a function of the concentration of compound 4 is shown. The data from one representative of several duplicate runs are shown. The kd values calculated from these curves (A and B) were given in .

Crystal structure of yCT and yCT-H9 complexed with compound 1.A, binding mode of compound 1 in yCT (green) crystal structure (RCSB entry code 1W2X) shows interactions of the two amide oxygens with the backbone NH of Glu-2026 and Gly-1958 as dashed lines. B, binding mode of compound 1 in the yCT-H9 (cyan) crystal structure, illustrating nearly identical interactions with the active site is shown. Note the shift in position of the terminal morpholine ring (foreground) by nearly 1 Å relative to its position in 1W2X. The structure of compound 1 (PDB code 1W2X) is depicted as green or cyan sticks.

Crystal structure of yCT and yCT-H9 complexed with compound 2.A, Compound 2 bound to yCT (green) crystal structure (RCSB entry code 3H0J) showing interactions of the two amide oxygens with the backbone NH of Glu-2026 and Gly-1958 as dashed lines. B, binding mode of compound 2 to yCT-H9 (cyan) crystal structure is shown. Note the altered orientation of the methylquinoline moiety, which has shifted by ∼180° relative to that seen in 3H0J. The structure of Compound 2 is depicted as green or cyan sticks.

Crystal structure of yCT and yCT-H9 complexed with compound 3.A, compound 3 bound to yCT (green) crystal structure (RCSB entry code 3H0Q). Interactions of the two amide oxygens with the backbone NH of Glu-2026 and Gly-1958 are shown as dashed lines. B, compound 3 bound to yCT-H9 (cyan) crystal structure. As in the case of compound 2, the methylquinoline is rotated by ∼180° between the yCT and yCT-H9 structures. The structure of compound 3 is depicted as green or cyan sticks.